The present invention provides nucleotide sequences from coryneform bacteria which code for the cysD, cysN, cysK, cysE and cysH genes and a process for the fermentative preparation of amino acids using bacteria in which the endogene genes mentioned are enhanced.
L-Amino acids, in particular L-lysine, L-cysteine and L-methionine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation procedures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and produce amino acids are obtained in this manner.
Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
However, there remains a need for improved methods of producing amino acids.
It is an object of the present invention to provide new methods for improved fermentative preparation of amino acids.
It is another object of the invention to provide nucleic acids which are useful for preparing amino acids.
Accordingly, the present invention provides isolated polynucleotides from coryneform bacteria comprising one or more of the polynucleotide sequences which code for the cysD gene, the cysN gene, the cysK gene, the cysE gene or the cysH gene, selected from the group consisting of
(a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
(b) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 3,
(c) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 5,
(d) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 6,
(e) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 8,
(f) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2,
(g) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 3,
(h) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 5,
(i) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 6,
(j) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 8,
(k) polynucleotide which is complementary to the polynucleotides of (a), (b), (c), (d), (e), (f), (g), (h), (i) or (j), and
(l) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k),
where the polypeptides preferably having the corresponding activities, namely of sulfate adenylyl transferase, cysteine synthase A, serine acetyl transferase or 3xe2x80x2-phosphoadenylyl sulfate reductase.
The present invention also provides the above-mentioned polynucleotides, these preferably being DNAs which are capable of replication, comprising:
(i) one or more nucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 4 or SEQ ID No. 7, or
(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
(iv) sense mutations of neutral function in (i).
The present invention also provides polynucleotides, in particular DNAs, which are capable of replication and comprise one or more nucleotide sequences as shown in SEQ ID No. 1, SEQ ID No.4, or SEQ ID No. 7;
polynucleotides which code for one or more polypeptides which comprises the corresponding amino acid sequences, as shown in SEQ ID No. 2, SEQ ID No.3, SEQ ID No. 5, SEQ ID No. 6, or SEQ ID No. 8;
a vector containing one or more of the polynucleotides according to the invention, in particular shuttle vectors or plasmid vectors, and
coryneform bacteria which contain the vector or in which one or more of the endogene genes chosen from the group consisting of the cysD gene, cysN gene, cysK gene, cysE gene and cysH gene is/are enhanced.
The present invention additionally provides a process for the fermentative preparation of amino acids using bacteria in which one or more endogene genes chosen from the group consisting of
the cysD gene which codes for the subunit II of sulfate adenylyltransferase,
the cysN gene which codes for the subunit I of sulfate adenylyl transferase,
the cysK gene which codes for cysteine synthase A,
the cysE gene which codes for serine acetyl transferase,
the cysH gene which codes for 3xe2x80x2-phosphoadenylyl sulfate reductase is enhanced.
All five endogene genes (cysD gene, cysN gene, cysK gene, cysE gene and cysH gene) participate in the biosynthesis of the sulfur-containing L-amino acids L-cysteine and L-methionine. The carbon matrix of these amino acids is predominantly derived from the same metabolic intermediates as that of the amino acids of the aspartate family, to which L-lysine belongs. Over-expression of one or more of the genes mentioned leads to pool shifts in the participating biosynthesis pathways, which has a positive effect on the formation of L-lysine, L-methionine and L-cysteine.
The present invention also provides a process for the fermentative preparation of an L-amino acid comprising:
(a) fermenting coryneform bacteria in a medium, wherein the bacteria produce the desired L-amino acid and in which at least the cysD gene, cysN gene, cysK gene, cysE gene and/or the cysH gene or nucleotide sequences which code for them is or are enhanced.
(b) concentrating the L-amino acid in the medium or in the cells of the bacteria, and
(c) isolating the L-amino acid.
The present invention further provides a process of the preparation of an L-methionine-containing animal feedstuffs additive from a fermentation broth, comprising:
(a) culturing and fermenting an L-methionine-producing microorganism in a fermentation medium;
(b) removing water from the L-methionine-containing fermentation broth;
(c) removing an amount of from 0 to 100 wt. % of the biomass formed during the fermentation; and
(d) drying he fermentation broth obtained according to (b) and/or (c) to obtain the animal feedstuffs additive in powder or granule form.
The present invention additionally provides A process of isolating nucleic acids, or polynucleotides or genes, such as RNA, cDNA, or DNA, which code for sulfate adenylyl transferase, cysteine synthase A, serine acetyl transferase and/or 3xe2x80x2-phosphoadenylyl sulfate reductase or have a high similarity with the sequences of the cysD gene, the cysN gene, the cysK gene, the cysE gene and/or the cysH gene, comprising:
contacting a sample with the polynucleotide described above under conditions such that the polynucleotide is capable of hybridizing to another polynucleotide which codes for sulfate adenylyl transferase, cysteine synthase A, serine acetyl transferase and/or 3xe2x80x2-phosphoadenylyl sulfate reductase or have a high similarity with the sequences of the cysD gene, the cysN gene, the cysK gene, the cysE gene and/or the cysH gene.
The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library of a coryneform bacterium, which comprises the complete gene or parts thereof, with a probe which comprises the sequence of the polynucleotides according to the invention according to SEQ ID No. 1, SEQ ID No.4 or SEQ ID No.7 or a fragment thereof, and isolation of the polynucleotide sequence mentioned.